Polycylic xanthones and their use

ABSTRACT

A novel class of antitumour compounds has been recognised based on the isolation from a new marine microbe, strain PO13-046, belonging to the genus Actinomadura sp.), of a compound designated IB-00208. The class of the formulae (I) or (II) where R 1  can be hydrogen, acyl, alkyl, alkenyl, aryl, benzyl, alkali metal, and/or sugar, and R 2  and R 3  can be hydrogen, alkyl, or together R 2  and R 3  form an unsaturated bond. Such compounds demonstrate an interesting activity several cancel cell lines and against Gram-positive bacteria.

BACKGROUND OF THE INVENTION

[0001] Cervinomycins (EP00246091; EP0054801; J.Antibiot. 1982, 35,645-652; J.Am.Chem.Soc. 1986, 108, 6088-6089, J.Antibiot. 1987, 40,301-308; J.Antibiot. 1994, 47, 342-348; synthetic derivativesJ.Antibiot. 1986, 39, 1636-1639 and EP0259496) and citreamicins(J.Antibiot. 1989, 42, 846-851; J.Antibiot. 1990, 43, 504-512;EP0405151) are members of a family of naturally occurring antibiotics,all of which posses a xanthone based unit embedded within a largerpolycyclic framework.

[0002] These compounds have demonstrated potent activity against aerobicand anaerobic bacteria and mycoplasma, but only cervinomycins have beendescribed to display antitumour activity (EP0246091).

[0003] We have recently described also the antitumour activity ofcitreamicins in PCT/GB01/02148.

[0004] New anticancer drugs are still needed for treatment against manyhuman tumours. Accordingly, a goal of the present invention is toprovide new antitumour agents with a polycyclic xanthone structure.

[0005] Another objective of this invention is to provide pharmaceuticalcompositions for administering to a patient in need of treatment anactive compound.

[0006] Yet another object is directed to the production of thepolycyclic xanthone by controlled aerobic fermentation using abiologically pure culture of an organism in appropriate nutrient media,also to provide with methods for its recovery and concentration from thefermentation broth, and to the final purification of the activecompound.

SUMMARY OF THE INVENTION

[0007] This invention provides a new class of active xanthone compoundsfounded on the discovery of a new compound IB-00208 isolated from abacteria, useful as antitumour medicaments with the formula:

[0008] Thus, the present invention provides compounds with the generalformula:

[0009] where each R¹ is the same or different and can be hydrogen, acyl,alkyl, alkenyl, aryl, benzyl, alkali metal, and/or sugar, and R² and R³can be hydrogen, alky, or together form an unsaturated bond.

[0010] This invention also provides processes for preparing suchcompounds including a process of obtaining IB-00208.

DETAILED DESCRIPTION OF THE INVENTION

[0011] IB-00208 and related compounds exhibits antitumour activityagainst mammalian tumours, such as human lung carcinoma, human coloncarcinoma, human melanoma, etc. Thus, the invention includes a method oftreating any mammal affected by a malignant tumour sensitive to them,which comprises administering to the affected individual atherapeutically effective amount of the compound or a pharmaceuticalcomposition thereof

[0012] The present invention also relates to pharmaceutical preparationswhich contain as active ingredient compound IB-00208 or any of itsderivatives, or a pharmaceutical acceptable salt thereof, as well as theprocesses for its preparation. A pharmaceutically acceptable carrier isemployed.

[0013] Pharmaceutical compositions are typically formulated from theactive compound and include any solid (tablets, pills, capsules,granules, etc.) or liquid (solutions, suspensions or emulsions) incombination with any carrier or other pharmacologically activecompounds.

[0014] The correct dosage of a pharmaceutical composition of IB-00208 orits derivatives will vary according to the particular compound,formulation, mode of application, and sites of host and tumour beingtreated.

[0015] Others factors like age, body weight, sex, diet, time ofadministration, rate of excretion, condition of the host, drugcombinations, reaction sensitivities and severity of the disease shallbe taken into account. Administration can be carried out continuously orperiodically within the maximum tolerated dose.

[0016] The acyl groups can be aliphatic acyl, aromatic acyl, or mixedaliphatic/aromatic acyl. Thus, for example, they can be of the formulaRCO-, where R is an alkyl group, an alkenyl group, an aryl group, anarylalkyl group, or an alkylaryl group. Examples include benzoyl.

[0017] The alkyl groups typically have from 1 to 18 carbon atoms. Alkylgroups preferably have from 1 to about 12 carbon atoms, more preferably1 to about 8 carbon atoms, still more preferably 1 to about 6 carbonatoms, and most preferably 1, 2, 3 or 4 carbon atoms. Methyl, ethyl andpropyl including isopropyl are particularly preferred alkyl groups inthe compounds of the present invention. As used herein, the term alkyl,unless otherwise modified, refers to both cyclic and noncyclic groups,although cyclic groups will comprise at least three carbon ring members.The alkyl groups may be straight chain or branched chain.

[0018] The alkenyl groups typically have from 1 to 18 carbon atoms.Preferred alkenyl groups in the compounds of the present invention haveone or more unsaturated linkages and from 2 to about 12 carbon atoms,more preferably 2 to about 8 carbon atoms, still more preferably 2 toabout 6 carbon atoms, even more preferably 2, 3 or 4 carbon atoms. Theterm alkenyl as used herein refer to both cyclic and noncyclic groups,although straight or branched noncyclic groups are generally morepreferred.

[0019] The aryl groups can be carbocyclic or heterocyclic, and may haveone or more fused rings. The carbocyclic aryl groups typically have 6 or10 carbon atoms, as in phenyl or naphthyl. Heterocyclic aryl groupstypically have 5 to 12 atoms, more usually 4, 5, 6, 10, 11 or 12 atoms,of which there is 1, 2, 3 or more heteroatoms usually chosen fromoxygen, sulphur or nitrogen. Suitable heterocyclic aryl groups in thecompounds of the present invention include coumarinyl including8-coumarinyl, quinolinyl including 8-quinolinyl, pyridyl, pyrazinyl,pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl,indolyl, benzofuranyl and benzothiazol.

[0020] Notable alkali metals include sodium or potassium.

[0021] Sugars employed as substituents are typically mono-, di- ortri-saccharides or saccharide derivatives, prepferably mono- ordi-saccharides. Pentose or hexose compounds are preferred. Derivativesinclude sugar glycosides, N-glycosylamines, O-acyl derivatives, O-methylderivatives, sugar alcohols, sugar acids, deoxy sugars, and relatedcompounds. Examples include the trimethyldeoxypyranose hexose ofIB-00208.

[0022] This invention describes a new polycyclic xanthone IB-00208,isolated from the fermentation broth of a microorganism, preferablyActinomadura sp. PO13-046, a culture of which has been deposited in theColección Española de Cultivos Tipo at the University of Valencia, Spainunder the accession number CECT 5318. This deposit has been made underthe provisions of the Budapest Treaty and all restrictions on theavailability thereof to the public will be made upon the granting of apatent on this application.

[0023] The microbial strain was isolated from an unidentified marinepolychaete collected at the Bay of Biscay.

[0024] While the deposited organism is clearly preferred, the presentinvention is not restricted or limited to any particular strain ororganisms. It is the intention of the present invention to include otherIB-00208 producing organisms, strains or mutants within the scope ofthis invention.

[0025] Actinomadura sp. PO13-046 cultured under controlled conditions ina suitable medium produces the antibiotic IB-00208. This strain ispreferably grown in an aqueous nutrient medium, under aerobic andmesophilic conditions.

[0026] The antibiotic IB-00208 can be isolated from the mycelial cake byextraction with a suitable mixture of solvent such as CHCl₃:CH₃OH:H₂O.The activity is concentrated in the lower layer. The extracts from tworepeated extractions can be combined and evaporated to dryness in vacuo.

[0027] Separation and purification of IB-00208 from the crude activeextract can be performed by the use of the proper combination ofconventional chromatographic techniques.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 is the proton NMR (¹H) spectrum of purified IB-00208;

[0029]FIG. 2 is the carbon-13 NMR (¹³C) spectrum of purified IB-00208;

[0030]FIG. 3 is the DEPT experiment of purified IB-00208;

[0031]FIGS. 4, 5, and 6 are COSY 45, HMQC and HMBC spectra of purifiedIB-00208 respectively;

[0032]FIG. 7 is the IR spectrum of purified IB-00208;

[0033]FIG. 8 is the HPLC/MS chromatogram and ESI-MS spectrum ofIB-00208; and

[0034]FIG. 9 is the HPLC/UV chromatogram and the UV spectrum of purifiedIB-00208

[0035] Fractionation can be guided by the antitumour activity offractions, or by TLC visualised with vanillin in conc. H₂SO₄, oranalytical HPLC with photodiode-array and MS detector. HPLC analysis isperformed at room temperature using an analytical column Symmetry C18 (5μm using as mobile phase methanol:H₂O:Acetic acid 99:1:1 and a flow rateof 0.3 ml/min. and plotted at 325 nm, in this conditions IB-00208retention time is 4.5 min as is shown in FIG. 9.

[0036] On the basis of detailed analysis of their various spectralcharacteristics, the pure compound can be identified as IB-00208 (seedata reproduced in FIGS. 1 to 9). Therefore the deduced structure is asit appears above.

[0037] IB-00208 data are summarised in Table 1. TABLE 1 UV spectrumabsorption maxima: 225, 255 nm and 325 nm as reported in FIG. 9. ¹H, ¹³Cand DEPT NMR. spectra are reported in FIG. 1, FIG. 2 and FIG. 3respectively. 2D NMR experiments COSY, HMQC and HMBC are reported inFIG. 4, FIG. 5 and FIG. 6 respectively. Infrared absorption spectrum isshown in FIG. 7. The ES-MS spectrum displayed a (M + H)⁻ peak at 691 and(M + Na) peak at 713, as reported in FIG. 8.

[0038] Various derivatives of IB-00208 can be prepared using techniquesalready known in the art. As an example, these modifications can be madeusing chemical or biological methods. These changes can include:

[0039] preparation of the aglycone moiety;

[0040] addition to the aglycone of different sugars known and new;

[0041] modification of the aglycone.

[0042] Compounds with a hydroquinone ring may be prepared from compoundswith quinone ring by reduction of the quinone with a reducing agent [forexample: NaBH4 (T.Ross Kelly et al. J.Am.Chem.Soc. 1989, 111,4522-4524), Na2S2O4 (A. V. Rama Rao et al. Tetrahedron Lett. 1991,32,5199-5202)] in an appropriate solvent, followed (when R1≠H) by reactingthe hydroquinone with an acylating or alkylating or appropriated agentin an appropriate solvent.

[0043] The present invention in reference to preferred embodiments willbe further illustrated with the following examples, which will aid inthe understanding of the present invention and are aimed only toillustrate it, but which are not to be construed as limitations thereof.

EXAMPLES OF THE INVENTION

[0044] All percentages reported herein, unless specified, are presentedby weight. All temperatures are expressed in degrees Celsius. Allincubations are carried out at 28° otherwise stated and flasks areshaken in an orbital shaker at 250 rpm. All media and recipients aresterile and all culture processes aseptic.

[0045] Examples 1 to 3 deal with the preparation of compound IB-00208with formula shown above, and Example 4 with its biological activity.

Example 1

[0046] The Producing Organism:

[0047] The taxonomic methods utilised herein are those usually employedin classic Actinomycete taxonomy and are reported in the literature.

[0048] All cultures after incubation were studied and records of resultswere made weekly up to 21 days. NaCl was added when needed.

[0049] A description of the organism is as follows:

[0050] After 21 days good growth was observed in ISP 2, ISP 6, Bennetand 172 ATCC with ASW. Colonies had light brown colour. In ISP 3, ISP 5,Czapek and ISP 7 with ASW less growth was obtained and no solublepigment was observed. No aerial mycelium was formed. Substrate myceliumwas branched. Isolated spores-over the substrate mycelium may occur.Spores are elongated and scarce. No other formations were observed.

[0051] In ISP-1 brown diffusible pigments were formed, as well as inother solid media. Resistance to NaCl was over 5%. The optimum growthtemperature range is between 25° and 35° C. The organism can grow onglucose, galactose, rhamnose, and xylose as the sole carbon source,however, growth on fructose, raffinose, m-inositol, sucrose, andα-melibiose is negative, and in mannitol and melezitose is doubtful.

[0052] Chemical composition studies of the organism show thatmeso-2,6-diaminopimelic acid is present in the whole cell hydrolysate ofstrain PO13-046.

[0053] Fatty acids methyl esters comparison of PO13-046 with othersimilar strains is described in TABLE 2. TABLE 2 i-14:0 14:0 15:0PO13-046 1.75 2.43 4.9 Actinomadura livida ATCC 33578 1.41 1.21 7.4Actinomadura madurae NRRL-B 5390 1.0 2.5 6.1 Actinomadura malachiticaATCC 27888 1.4 3.4 0.7 Actinomadura vinacea ATCC 33581 1.4 1.4 6.1Actinomadura formosensis ¹ ATCC 49059 1.3 1.2 2.8 Streptomyces griseus*DSM 40236 15.1 0.8 0.9 i-16:0 16:1 16:0 PO13-046 20.2 5.16 22.6Actinomadura livida ATCC 33578 21.8 5.5 13.7 Actinomadura madurae NRRL-B5390 11.5 5.56 21.2 Actinomadura malachitica ATCC 27888 18.5 7.9 26.2Actinomadura vinacea ATCC 33581 22.7 6.8 15.2 Actinomadura formosensis ¹ATCC 49059 22.5 3.4 17.8 Streptomyces griseus* DSM 40236 21.2 5.1 6.417:1 17:0 PO13-046 6.5 5.4 Actinomadura livida ATCC 33578 14.9 7.0Actinomadura madurae NRRL-B 5390 10.9 7.9 Act. malachitica ATCC 278881.1 0.9 Actinomadura vinacea ATCC 33581 12.8 5.6 Actinomaduraformosensis ¹ ATCC 49059 6.9 4.6 Streptomyces griseus* DSM 40236 <1 <1i- Cis- i-18:1 18:0 18:1 18:0 18.38 PO13-046 5.2 1.8 13.0 1.22 5.75Actinomadura livida ATCC 33578 7.5 1.2 7.8 1.0 5.7 Actinomadura maduraeNRRL- 3.0 1.6 16.5 1.1 5.3 B 5390 Act. malachitica ATCC 27888 3.7 2.715.8 4.5 8.7 Actinomadura vinacea ATCC 33581 6.1 1.6 10.3 0.7 5.1Actinomadura formosensis ¹ 6.8 1.8 10.8 2.4 5.5 ATCC 49059 Streptomycesgriseus* DSM 40236 1.1 <1 <1 <1 <1

[0054] Based on the preceding characteristics the culture is determinedas a species of the genus Actinomadura, with 94% similarity to A.vinacea type strain.

Example 2

[0055] Fermentation of IB-00208:

[0056] The required steps needed for the production of IB-00208 by thepreferred organism are:

[0057] Stock Culture: Whole broth of a pure culture of Actinomadura sp.strain PO13-046 is preserved frozen in 20% glycerol.

[0058] Inoculum: A frozen culture or a well grown slant culture is usedto seed 100 ml of seed medium described previously in a 250 cc shakeflask. The flask is incubated during 48 hrs, and used as a first stageinoculum. 500 ml of the same medium in 2 L Erlenmeyer flask are seededwith 10% of this first stage inoculum. The flask is incubated during 48h.

[0059] Fermentation: With 2.5L of second stage inoculum seed 50 L ofproduction medium already described in a 75 L fermentation tank. Thefermentation is carried out during 96 hours with 400 rpm agitation andair flow of 0.5 V/V.M. TABLE 3 Inoculum medium: Glucose 5 g Starch 20 gBeef extract 3 g Yeast extract 5 g Tryptone 5 g CaCO3 4 g NaCl 5 g KCl0.5 g MgCl2 2 g Tap water to 1,000 ml Production medium: Glucose 5 gStarch 20 g Soybean meal 15 g Yeast extract 5 g Tryptone 2 g CaCO3 4 gNaCl 5 g KCl 0.5 g MgCl₂ 2 g Tap water to 1,000 ml

[0060] Yield of IB-00208 can be monitored by whole broth assay againstmurine leukaemia P-388 or by HPLC.

Example 3

[0061] Isolation of IB-00208:

[0062] 4.5 litres of whole harvested broth were filtrated to separatethe biomass and other solids. The mycelia cake was extracted twice witha mixture solvent (1.5 l) of CHCl₃:CH₃OH:H₂O (2:1:1), the activity wasconcentrated in the lower layer. The organic solvent was concentratedand evaporated to dryness in vacuo to yield 1.2 g of crude extract.

[0063] The extract was chromatographed on silica gel using a mixture ofn-hexane/ethyl acetate and ethyl acetate/methanol as eluting solvents.110 mg of a fraction containing IB-00208 with antitumour activity wereeluted with ethyl acetate/methanol 1:1.

[0064] Further purification of the fraction containing IB-00208 wasachieved by column chromatography on silica gel and 18 mg of purecompound IB-00208 were eluted with chloroform/methanol 95:5.

Example 4

[0065] Biological activity:

[0066] The antitumour activities of IB-00208 have been determined invitro in cell cultures of mouse leukaemia P-388, human lung carcinomaA-549, human colon carcinoma HT-29 and human melanoma MEL-28. Theprocedure was carried out using the methodology described by Bergeron etal. The IC50 found was of 0.001, 0.001, 0.001, and 0.001 μg/ml for all 4cell lines.

REFERENCES

[0067] The following references have been cited herein:

[0068] ATCC Catalog 1996.

[0069] Bergeron et al. Biochem.Biophys. Res.Comm. 121:848, 1984

[0070] Guerrant and Moss, Anal.Chem. 56:633, 1984.

[0071] Hasegawa et al., J. Gen. Appl. Microbiol. 29:319, 1983.

[0072] Shirling and Gotlieb. Int.J.Syst.Bacteriol. 16:313, 1966.

[0073] Van der Auwera et al., J. Microbiol. Methods, 4:265, 1986.

[0074] Waksman, The Actinomycetes vol.II:331, 1961.

[0075] The present invention has been described in detail, including thepreferred embodiments thereof. However, it will be appreciated thatthose skilled in the art, upon consideration of the present disclosure,may make modifications and/or improvements within this invention.

1. A compound of the general formula:

where each R¹ is the same or different and can be hydrogen, acyl, alkyl,alkenyl, aryl, benzyl, alkali metal, and/or sugar, and R² and R³ can behydrogen, alkyl, or together R² and R³ form an unsaturated bond.
 2. Acompound according to claim 1 of formula:


3. A process of producing the compound IB-00208, which comprisescultivating a strain of a microorganism capable of producing it in anaqueous nutrient medium.
 4. The process according to claim 3, whichfurther comprises isolating and purifying compound IB-00208 from thecultured broth.
 5. The process according to claim 4, wherein theIB-00208 producing organism is the substantially pure culture strainPO13-046, available under accession number CECT 5318, from the ColecciónEspañola de Cultivos Tipo at the University of Valencia, Spain.
 6. Asubstantially pure culture strain PO13-046 as isolated from marineinvertebrates, which belongs to the family Thermomonosporaceae, and hasbeen taxonomically classified as Actinomadura sp.
 7. An anti-tumourpharmaceutical composition comprising a compound according to claim 1and a pharmaceutically acceptable carrier.
 8. An anti-tumourpharmaceutical composition comprising the compound IB-00208 according toclaim 2, and a pharmaceutically acceptable carrier.